EP1523562B1 - Expression von rezeptoren mit 7 transmembrandomänen in einem baculovirus/insektenzellen system - Google Patents

Expression von rezeptoren mit 7 transmembrandomänen in einem baculovirus/insektenzellen system Download PDF

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EP1523562B1
EP1523562B1 EP03750832A EP03750832A EP1523562B1 EP 1523562 B1 EP1523562 B1 EP 1523562B1 EP 03750832 A EP03750832 A EP 03750832A EP 03750832 A EP03750832 A EP 03750832A EP 1523562 B1 EP1523562 B1 EP 1523562B1
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baculovirus
promoter
protein
receptors
receptor
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French (fr)
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EP1523562A1 (de
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Gérard Devauchelle
Jacques Demaille
Conception Ferraz
Valéry MATARAZZO
Catherine Ronin
Martine Cerutti
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Centre National de la Recherche Scientifique CNRS
Universite de Montpellier
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Centre National de la Recherche Scientifique CNRS
Universite de Montpellier
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the invention relates to the production, in a baculovirus system / insect cells, seven-fold receptors transmembrane domains, including receptors coupled to G proteins.
  • the super-family of seven-domain receivers transmembrane protein G includes including many membrane receptors of neurotransmitters, neuropeptides and hormones. they have all the same structural organization, namely a single polypeptide chain comprising seven hydrophobic domains which cross the membrane double lipid layer.
  • the olfactory receptors represent a very large family and constitute in vertebrates the numerically most important family (ZOZULYA et al., Genome Biology, 6 (2), 2001: http: // www.genomebiology.com/2001/2/6/research/0018.1 , ZHANG and FIRESTEIN, Nat Neurosci., 5 (2), 124-133, 2002). They are mainly located on the surface of neuroreceptor cell membranes (olfactory neurons) of the olfactory epithelium, but their expression in other tissues has also been reported.
  • CAMP MPAs cyclic
  • IP 3 inositol triphosphate
  • G proteins that have been identified as implicated in olfactory signal transduction by the cAMP pathway are G ⁇ s and G ⁇ olf.
  • Go or Gq proteins FDOOL et al ., Chemical Senses, 20, 489-498, 1995, SCHANDAR et al ., J. Biol Chem. , 273, 16669-16677, 1998) could be involved.
  • G protein-coupled receptors have been expressed in functional form in different heterologous systems: the baculovirus / insect cell system, which has been used, for example, to express adrenergic receptors, muscarinic receptors, serotonin receptors, etc. canabinoids, oxytocin, substance P, etc.
  • heterologous system is of a particular interest in the case of receivers olfactory, which are mostly orphan receptors, whose ligands are not known.
  • olfactory receptors have been expressed in mammalian or insect cells.
  • RAMING et al. (Nature, 361 (3410), 353-356, 1993) expressed rat OR5 receptor in Sf9 cells after recombinant baculovirus infection, and observed transient increases in second messenger IP 3 in infected cells in response to stimulation by lyral or lilial. Similar results have been reported by BREER et al . (Annals of the New York Academy of Sciences, 855, 175-181, 1998) and GAT et al. (European Journal of Biochemistry, 225 (3), 1157-1168, 1994).
  • Olfactory receptors in mature olfactory neurons, in order to allow on the one hand their addressing and insertion correct in the plasma membrane, and secondly provide a system of suitable second messengers capable of generate a signal large enough to be detected (U.S. Patent 5,993,778).
  • the inventors have now developed a system improving the expression of receptors coupled to G proteins, including olfactory receptors, in insect cells.
  • a promoter is used in which the deleted portion includes at least the region extending from positions -1 to -5 compared to the initiation site of the translation of polyhedrin.
  • said expression cassette comprises in in addition, upstream of the sequence b), a coding sequence c) for a signal peptide.
  • sequences coding for signal peptides usable for the implementation of of the present invention, mention may be made of the signal peptide Ecdysteroid UDP Glucosyl Transferase (EGT) from baculovirus AcNPV, the signal peptide of lactotransferrin cattle, etc.
  • EGT Ecdysteroid UDP Glucosyl Transferase
  • Said expression cassette can also understand upstream or downstream of the sequence b) coding for the receptor with seven transmembrane domains, a sequence d) coding for a label peptide facilitating detection of the recombinant receptor expressed.
  • sequence b) coding for the receptor with seven transmembrane domains a sequence d) coding for a label peptide facilitating detection of the recombinant receptor expressed.
  • An expression cassette according to the invention can be built to express any receiver to seven transmembrane domains.
  • said receptor is an olfactory receptor.
  • the present invention also relates to a method for expressing a seven-domain receiver transmembrane in an insect cell, characterized in that what one infects said insect cell with a recombinant baculovirus comprising an expression cassette as defined above.
  • said cassette expression is inserted in place of the sponsor and the native gene of the polyhedrin said baculovirus.
  • a G protein that can be labeled with its N-terminus by a peptide tag, for example the HA epitope, without interfering with the expression of the protein on the surface of the insect cell or its coupling to the receiver.
  • said protein G is expressed under the control of the P10 gene promoter of a baculovirus.
  • the inventors have indeed found that the use of the P10 gene promoter, the expression of which is a little earlier than that of the polyhedrin gene, allows to express a receiver to seven domains transmembrane in a cell already containing a important amount of protein G, and thus to optimize the coupling between said receptor and said protein.
  • Co-expression of the seven-domain receptor transmembrane and of said protein G can be performed by co-infecting an insect cell with two baculoviruses recombinants: one expressing the receptor in a cassette expression according to the invention, and the other expressing said protein G under the control of the protein promoter P10.
  • the present invention also relates to recombinant vectors carrying at least one cassette expression according to the invention as defined above.
  • the present invention also relates to insect cells infected with a recombinant baculovirus according to the invention; these cells express to their receptor surface with seven transmembrane domains functional, capable of coupling to either G proteins endogenous baculoviruses, in the case where they are expressed alone, or to the exogenous G proteins coexpressed in the same cell.
  • Insect cells according to this invention can be used for the study and the characterization of seven-domain receptors transmembrane, especially to verify functionality putative receptors, identify receptor ligands orphans, or study mechanisms involved in receptor activation, signal transduction, by example the identification of the protein (s) G partner (s) concerned, the study of the coupling to these proteins, and the function of the complex thus formed, etc ..
  • the signal resulting from the binding of these receptors with their ligand (s) can be detected by different techniques that are known in themselves.
  • variations of calcium intracellular including calcium imaging, or variations of second messengers, inositol phosphate or AMP cyclic; we can also detect phosphorylation and / or the kinase activity of the proteins involved in the transduction cascade, etc.
  • insect cells according to the invention for expressing receptors with seven mutant transmembrane domains, and screen the mutant (s) recognizing a ligand of interest.
  • insect cells according to the present invention are used as described above, for the study and characterization of olfactory receptors.
  • the ligands of the olfactory receptors can be odorant molecules, proteins transporting (OBP for: "olfactory binding proteins"), odorous molecule / OBP complexes, or molecules synthetic materials such as cyclodextrins used in perfumery (soaps, candles, papers, etc.) as fixatives odorants very gradually releasing the volatile odorant.
  • OBP proteins transporting
  • odorous molecule / OBP complexes or molecules synthetic materials such as cyclodextrins used in perfumery (soaps, candles, papers, etc.) as fixatives odorants very gradually releasing the volatile odorant.
  • insect cells that comply with the invention expressing said receptor and a G protein partner of said receptor to identify the ligand (s) the receiver concerned, testing separately the response of that receptor with different odorous or complex molecules odorous molecule / OBP.
  • insect cells according to the invention expressing functional olfactory receptors and G proteins partners of these receptors to identify those who respond to a ligand of interest, testing separately the response of each of these receptors to different molecules odorous or complex odor molecule / OBP.
  • Insect cells according to the invention expressing a functional olfactory receptor whose ligand has have been identified can be advantageously used for obtaining biosensors, allowing the detection of odorant molecules, especially for analysis and control quality of volatile components, which include aromatic products of interest, and / or for the detection products likely to affect the organoleptic qualities, or for the detection of potentially harmful products.
  • EXAMPLE 1 OBTAINING RECOMBINANT BACULOVIRUS EXPRESSING HUMAN OLFACTORY RECEPTORS UNDER THE CONTROL OF A PROMOTER MODIFIED POLYEDRINE.
  • oligonucleotide is inserted into the vector pGmAc 217, immediately downstream of the modified polh promoter.
  • the sequence of this oligonucleotide is as follows:
  • This oligonucleotide comprises a sequence of 54 bp (underlined) coding for the signal peptide of Ecdysteroid UDP Glucosyl Transferase (EGT) baculovirus AcNPV (GenBank M22619), followed by the coding sequence for the FLAG epitope (in italics), followed by two sites unique NcoI and KpnI restriction (in bold).
  • S and AS primers are used:
  • Amplifications are performed in the following reaction mixture:
  • Both PCR products are digested with NcoI / KpnI and the resulting fragments are subcloned between the NcoI and KpnI sites of the vector pGmAc 217 containing the signal peptide.
  • Recombinant viruses are obtained by co-transfection of Sf9 cells with baculovirus DNA wild AcMNPV, and pGmAc transfer vector DNA 217-209 or pGmAc 217-210.
  • Sf9 cells of Spodoptera frugiperda are cultured in plastic bottles of 25 or 75 cm 2 and maintained at 28 ° C. in a TC100 medium (GIBCO-BRL) containing 5% fetal bovine serum (GIBCO-BRL). Subcultures are done twice a week.
  • Wild-type baculovirus DNA AcMNPV is prepared from viral particles.
  • Co-transfection is performed by lipofection, by incubating 2 ⁇ 10 6 cells with 3 ml of serum-free medium containing 500 ng of baculovirus DNA and 10 ⁇ g of transfer vector, mixed with 40 ⁇ l of DOTAP (ROCHE, France). . After 4 hours of incubation at 28 ° C., the transfection mixture is removed and replaced with 5 ml of serum-supplemented medium. The recombinant baculoviruses are recovered from the supernatant of the transfected cells after 5 days of incubation at 28 ° C.
  • Recombinant baculoviruses are isolated by three purification steps per lysis range and selected on the basis of a phenotype ob- (absence of body of inclusion), then multiplied by cellular infections successive.
  • the recombinant baculoviruses referred to hereinafter AcMNPV209 and AcMNPV210 respectively contain the sequence encoding the OR-209 receptor and the coding sequence for the OR-210 receiver.
  • Figure 1 A represents a general diagram of different stages of building a baculovirus recombinant comprising a coding sequence for a receptor OR, associated with the signal peptide of the EGT gene and the marking of the FLAG epitope.
  • Baculovirus AcMNPV209 and AcMNPv210 are shown in Figure 1C.
  • EXAMPLE 2 OBTAINING A DOUBLE-RECOMBINANT BACULOVIRUS EXPRESSING A HUMAN OLFACTORY RECEPTOR UNDER CONTROL OF A PROMOTER OF MODIFIED POLYEDRINE, AND PROTEIN G UNDER MONITORING THE PROMOTER P10.
  • the vector p119 (LEMEULLE et al., FEBS is used Lett., 423, 159-166, 1998) containing the cloning sites single BglII and HindIII located downstream of the p10 promoter of baculovirus.
  • the cDNA encoding the ⁇ 16 G protein was obtained from a cDNA library of the promyelocytic line of human leukemia HL60 (CLONTECH, France).
  • the labeling sequence by the HA epitope is introduced by PCR.
  • S and AS sense and antisense oligonucleotides
  • Amplification is carried out in the mixture following reaction:
  • the cDNA encoding the ⁇ olf G protein was cloned by RT-PCR from a total brain total RNA preparation provided by Dr. Gilles TOUMANIANTZ (IPMC, Nice, France).
  • the mRNAs are obtained by incubating (5 min at 70 ° C. and 5 min at 4 ° C.) 4 ⁇ g of total RNA with 1 ⁇ g of oligonucleotide primer dT (INVITROGEN) in a final volume of 15 ⁇ l. 10 ⁇ l of the mRNA preparation are then used as a sample for reverse transcription into cDNA using the "cDNA cycle" kit from INVITROGEN (Netherlands). Retro-transcription is performed at 42 ° C for 70 min and the reaction is inactivated at 94 ° C for 5 min. The synthesized cDNA is used as a template for subsequent PCR amplifications.
  • the labeling sequence by the HA epitope is introduced by PCR.
  • S and AS antisense oligonucleotides used for this PCR are as follows:
  • the PCR amplification conditions are identical to those used for G ⁇ 16 .
  • Both PCR products are digested with BglII / HindIII and the resulting fragments are subcloned between the BglII and HindIII sites of the p119 vector.
  • the transfer vectors obtained designated p119-HA- G ⁇ 16 and p119-HA-G olf pGmAc 217-209 respectively contain the sequence encoding HA-G ⁇ 16 , and the coding sequence for HA-G olf .
  • Sf9 Spodoptera frugiperda cells cultured as described in Example 1 above were co-transfected with baculovirus DNA AcSLP10, and one of the vectors p119-HA-16 or G-HA-p119 G olf.
  • Baculovirus AcSLP10 (CHAABIHI et al., J. Virol., 67, 2664-2671, 1993) is a modified baculovirus that has a single strong late promoter (P10), which controls expression of the polyhedrin coding sequence.
  • Co-transfection is performed by lipofection, as described in Example 1 above.
  • the recombinant baculoviruses obtained, called AcSLP10G 16 and AcSLP10G olf respectively express p119-HA-G 16 or p119-HA-G olf , under the control of the p10 promoter. These baculoviruses are multiplied as described in Example 1 above.
  • Double-recombinant baculoviruses Double-recombinant baculoviruses.
  • Cells Sf9 Spodoptera frugiperda are co-transfected with baculovirus DNA AcSLP10, and one of p119-HA-16 or G-HA-p119 G olf vectors and the transfer vector pGmAcI50 (DEVAUCHELLE and CERUTTI, the Insect Baculovirus: Foreign Gene Expression Vectors, Focus on Biochemistry, No. 5, C2, 1991) which contains the coding sequence for polyhedrin under the control of its own promoter.
  • the recombinant baculoviruses expressing G proteins and polyhedrin are selected on the basis of their ob + phenotype (presence of inclusion bodies), and multiplied.
  • the recombinant baculoviruses obtained, expressing respectively p119-HA-G 16 or p119-HA-G olf , under the control of the p10 promoter, are called AcSLP10G 16 Ph and AcSLP10G olf Ph.
  • each AcSLP10G 16 Ph or AcSLP10G olf Ph baculovirus is used to co-transfect Sf9 cells with baculovirus pGmAc 217-209 or baculovirus pGmAc 217-210.
  • the double-recombinant baculoviruses obtained are selected on the basis of an ob-viral phenotype. These recombinant baculovirus double are referred to as AcG 16 -209; AcG olf -210; AcG 16 -209; AcG olf -210.
  • FIG. 1B represents a general diagram of the different steps of construction of a recombinant double baculovirus comprising a sequence encoding a G protein associated with the labeling sequence of the HA epitope under the control of the P10 promoter, and a sequence coding for a OR receptor, associated with EGT signal peptide and FLAG epitope tagging sequence, under the control of the modified polyhedrin gene promoter.
  • Baculoviruses AcG 16 -209, AcG olf -210, -209 and AcG 16 AcG olf -210 are shown in Figure 1 C.
  • EXAMPLE 3 EXPRESSION OF OLFACTORY RECEPTORS AND G PROTEINS IN INSECT CELLS.
  • Sf9 cells are harvested 36-48 hours after infection.
  • the production of olfactory receptors or recombinant G protein is evaluated by transfer immunoelectrophoretic, using the directed antibodies against their FLAG or HA epitope tag.
  • Cells infected with baculovirus AcMNPV209 and AcMNPV210 express proteins recognized by the anti-FLAG antibody, and of corresponding molecular weight to that of the ORs.
  • the cells infected with baculovirus AcSLP10G 16 and AcSLP10G olf express proteins recognized by the anti-HA antibody, and of molecular weight corresponding to that of the G proteins.
  • Sf9 cells are grown on 24-well plates containing 12 mm coverslips. Before the experiments, the culture medium is removed and washed with a buffer containing 10 mM NaCl, 60 mM KCl, 25 mM MgCl 2 , 1.8 mM CaCl 2 , 4 mM D-glucose, 110 mM sucrose and 10 mM 2- (N-morpholino) ethanesulfonic acid; the pH is adjusted to 6.2 at room temperature with the TRIZMA base.
  • the cells are incubated for 1 hour at Ambient temperature in the dark with MBS solution containing 2-AM fura (4 ⁇ M) (molecular probe). The cells are then washed with an excess of MBS without fura 2-AM and mounted in a COVERWELL room (POLYLABO, France) (diameter: 9 mm, thickness: 2.5 mm, section: 22.5).
  • MBS solution containing 2-AM fura (4 ⁇ M) (molecular probe).
  • the cells are then washed with an excess of MBS without fura 2-AM and mounted in a COVERWELL room (POLYLABO, France) (diameter: 9 mm, thickness: 2.5 mm, section: 22.5).
  • the chamber is placed under the microscope, and the cells are infused under gravity with 0.85 ml / min (3.78 cm / min) at room temperature (23 ° C).
  • the fura-2 fluorescence measurements are made using a multi-path wavelength illumination system POLYCHROME II TILL PHOTONICS GmbH, (PLANEGG) for quenching.
  • the microscope (OLYMPUS) is equipped with a long range distance lens (X20).
  • Spatio-temporal distributions of Ca 2+ are studied using a PCO interlaced camera.
  • the excitation wavelength of the dye varies alternately between 340 and 380 nm and is adapted to the exposure time of the camera (20-40 msec, binning: 2).
  • the acquisition and calculation of the fluorescence images are performed using the TILL-VISION software. All signals are related to background noise. Fluorescence ratios (f 340 / f 380 ) are calculated for intracellular Ca 2+ variations.

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Claims (15)

  1. Expressionskassette, umfassend:
    a) einen Promoter, der vom Promoter des Polyhedrins eines Baculovirus durch Deletion der ganzen Region oder eines Teils der Region des Promoters, die sich von Position-1 bis Position -12 bezüglich der Initiationsstelle der Translation des Polyhedrins erstreckt, abgeleitet ist;
    b) eine Sequenz, die für einen Rezeptor mit sieben Transmembrandomänen codiert, die unter der Transkriptionskontrolle des genannten Promoters steht.
  2. Expressionskassette nach Anspruch 1, dadurch gekennzeichnet, dass sie außerdem stromaufwärts zu Sequenz b) eine Sequenz, die für ein Peptidsignal codiert, umfasst.
  3. Expressionskassette nach einem der Ansprüche 1 oder 2, dadurch gekennzeichnet, dass der genannte Rezeptor mit sieben Transmembrandomänen ein olfaktiver Rezeptor ist.
  4. Verfahren zum Expremieren eines Rezeptors mit sieben Transmembrandomänen in einer Insektenzelle, dadurch gekennzeichnet, dass man die Insektenzelle mit einem rekombinanten Baculovirus infiziert, das eine Expressionskassette nach einem der Ansprüche 1 bis 3 umfasst.
  5. Verfahren nach Anspruch 4, dadurch gekennzeichnet, dass man in derselben Insektenzelle auch ein Protein G exprimiert.
  6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, dass das Protein G unter der Kontrolle des Promoters des Gens P10 eines Baculovirus exprimiert wird.
  7. Verfahren nach Anspruch 6, dadurch gekennzeichnet, dass man ein doppelt rekombinantes Baculovirus verwendet, umfassend:
    eine Expressionskassette nach einem der Ansprüche 1 bis 3 und
    eine Sequenz, die für ein Protein G kodiert, die unter der Transkriptionskontrolle des Promoters des Gens P10 des genannten Baculovirus steht.
  8. Rekombinantes Baculovirus, das eine Expressionskassette nach einem der Ansprüche 1 bis 3 umfasst.
  9. Rekombinantes Baculovirus nach Anspruch 8, dadurch gekennzeichnet, dass die Expressionskassette als Ersatz für den Promoter und das Gen des Polyhedrin des Baculovirus insertiert ist.
  10. Rekombinantes Baculovirus nach Anspruch 9, dadurch gekennzeichnet, dass das Baculovirus außerdem eine Sequenz umfasst, die für ein Protein G kodiert, die unter die Transkriptionskontrolle des Promoters des Gens P10 gestellt ist.
  11. Insektenzelle, die durch ein rekombinantes Baculovirus nach einem der Ansprüche 8 bis 10 infiziert ist.
  12. Verwendung einer Insektenzelle nach Anspruch 11 zur Bestimmung der Funktionalität eines vermeintlichen Rezeptors mit sieben Transmembrandomänen.
  13. Verwendung einer Insektenzelle nach Anspruch 11 zur Identifizierung des Liganden (der Liganden) eines orphanen (verwaisten) Rezeptors mit sieben Transmembrandomänen.
  14. Verwendung einer Insektenzelle nach Anspruch 11 zur Identifizierung eines Rezeptors (von Rezeptoren) mit sieben Transmembrandomänen, die fähig sind, an einen Liganden von Interesse zu binden.
  15. Verwendung nach einem der Ansprüche 12 bis 14, dadurch gekennzeichnet, dass der Rezeptor mit sieben Transmembrandomänen ein olfaktiver Rezeptor ist.
EP03750832A 2002-07-24 2003-07-22 Expression von rezeptoren mit 7 transmembrandomänen in einem baculovirus/insektenzellen system Expired - Lifetime EP1523562B1 (de)

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FR0209377A FR2842824B1 (fr) 2002-07-24 2002-07-24 Expression de recepteurs a 7 domaines transmembranaires en systeme baculovirus/cellules d'insectes
FR0209377 2002-07-24
PCT/FR2003/002309 WO2004011658A1 (fr) 2002-07-24 2003-07-22 Expression de recepteurs a 7 domaines transmembranaires en systeme baculovirus/cellules d'insectes.

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CN109071626A (zh) 2016-02-24 2018-12-21 阿罗姆斯公司 用于检测气味、香味和味道的生物传感器
US10209239B1 (en) 2017-08-16 2019-02-19 Aromyx Corporation Method of making an aromagraph comprising ectopic olfactory receptors

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US5879680A (en) * 1987-12-23 1999-03-09 The United States Of America As Represented By The Department Of Health And Human Services Cloned DNA for synthesizing unique glucocerebrosidase
US5236838A (en) * 1988-12-23 1993-08-17 Genzyme Corporation Enzymatically active recombinant glucocerebrosidase
US5516657A (en) * 1992-05-11 1996-05-14 Cambridge Biotech Corporation Baculovirus vectors for expression of secretory and membrane-bound proteins
JP4429385B2 (ja) * 1994-07-15 2010-03-10 セフアロン・インコーポレーテツド バキュロウイルスにより発現される活性カルパイン
US6433156B1 (en) * 1998-12-22 2002-08-13 Smithkline Beecham Corporation Polynucleotide and polypeptide sequence of rabbit G-protein alpha 16

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AU2003269045A8 (en) 2004-02-16
EP1523562A1 (de) 2005-04-20
CA2492977A1 (fr) 2004-02-05
US20060009618A1 (en) 2006-01-12
AU2003269045A1 (en) 2004-02-16
FR2842824B1 (fr) 2004-11-12
DE60301889D1 (de) 2006-02-23
ATE306554T1 (de) 2005-10-15
FR2842824A1 (fr) 2004-01-30

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